The packaging of the genome into chromatin is essential for normal growth, development, and differentiation. Nucleosomes are the basic repeating unit of chromatin that stabilize and restrict access to the DNA, forming a dynamic structure that tightly regulates all of the processes that use DNA as a substrate, including transcription, DNA replication, DNA repair, and recombination. Nucleosome assembly and disassembly processes are important in human disease, as seen in the multiple genetic malformations and cancers that have been linked to aberrations in proteins that form and modify chromatin structure. The key proteins responsible for replication-dependent nucleosome assembly of histone H3 and H4 are the H3/H4 histone chaperones, Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor (CAF-1), as well as proliferating-cell nuclear antigen (PCNA), which targets these to sites of newly replicated DNA. These proteins are highly conserved throughout eukaryotic evolution and are the focus of this study because of their central role in histone H3/H4 deposition onto newly replicated DNA. Our biophysical and structural studies have revealed unexpected and interesting insights into the early stages of replication-dependent chromatin assembly, namely the hand-off mechanism involving transfer of dimers of H3/H4 from Asf1 to CAF-1. The Asf1-H3/H4 complex comprises one molecule of Asf1 bound to an H3/H4 heterodimer through the H3 dimerization interface and the C-terminus of H4. We have recently found that CAF-1 has a unique mechanism for carrying H3/H4 as an asymmetric non-canonical H3/H4 tetramer. In Aim 1, will delineate the interactions that are involved in the formation of CAF-1 and CAF-1 H3/H4 complexes. Aim 2 addresses the intrinsic thermodynamic properties of the recruitment of H3/H4 to DNA via interactions with CAF-1 and PCNA-loaded DNA. These studies take advantage of recombinant chromatin assembly factors, novel biophysical and structural approaches that have been specifically developed to study histone chaperones, as well as physiological analyses in the yeast model system. This work will elucidate a new mechanism for H3/H4 chaperone activity and explain the fundamental basis of H3/H4 recruitment to DNA via CAF-1 and PCNA.